摘要
对插入质粒pUC18－181上的微紫青霉（Penicilliumjanthinellum）CBHI酶的cDNA基因进行一系列DNA体外操作，包括进行序列定向缺失，最后将两末端修饰为平端后进行连接使质粒环化。用得到的产生序列定向缺失的重组质粒转化大肠杆菌JM109。利用CBD能吸附到结晶纤维素上的特性，从随机选取的24个缺失转化子中筛选到一株含CBD编码区的转化子JM109（pUC18C），所表达的CBD融合蛋白分子量为21kD．JM109（pUC18C）所产生的LacZ-CBD融合蛋白可通过对纤维素的吸附-解吸附过程一步纯化。其IPTG诱导的pNPC酶活力为零，表明该菌已不再具有CBHI酶活力。
The in vitro DNA manipulations,included the nested deletions,of cbh1 from p.janthinellum inserted into pUC18-181 were carried out. The two ends of fragments were modified into blunt ends and the fragments were self-ligated. Then,the encircled plasmids were transformed to E.colt JM109.Utilizing the characterization of CBD binding to crystalline cellulose,one catalytic domain deletion transformant producing active LacZ-CBD fusion protein was isolated from 24 transformants randomly Picked from 400 transformants.The molecular weight of the LacZ-CBD fuSion protein is 21 kD. The plasmid was designated pUC 18C.The LacZ-CBD fusion protein produced by JM109(pUC18C)was able to be Purified by procedure of adsorption-desorption to cellulose.The pNPC activity of crude enzyme solution of JM109(pUC18C) induced by IPTG were zero,idenhfied that JM109(pUC18C)has no CBHI activity.
出处
《微生物学报》
CAS
CSCD
北大核心
1998年第4期269-275,共7页
Acta Microbiologica Sinica
基金
国家自然科学基金
山东省自然科学基金
国家教委博士点基金
关键词
大肠杆菌
亚克隆
表达
CBHI酶
微紫青霉
Nested set of deletion,Cellobiohydrolase I,Cellulose binding domain,Penicillium janthinellum
